Enzyme Binding-Inhibiting Assay for Iodothyronine 5′-Monodeiodinase (5′-MD) and Its Application to Isolation of Complementary Deoxyribonucleic Acid Clones for the 5′-MD in Rat Liver

Abstract

To identify and/or quantify type I T4 5''-monodeiodinase (5''-MD) immunologically, polyclonal antibodies were produced by immunization of rabbits with solubilized microsomal proteins (SMP) from rat liver. Pilot studies showed that the antibody binds to, but does not neutralize, rat liver enzyme. We have employed the polyclonal antibody to develop a 5''-MD enzyme binding-inhibiting assay (MBIA). For this purpose, active, inactive, or synthetic 5''-MD was preincubated with rabbit antibody and removed by Staphylococcus aureus protein-A (Staph-A). The 5''-MD-binding sites that were left on the Staph-A-bound rabbit antibody were assayed by adding active 5''-MD in fresh liver SMP. After centrifugation of Staph-A, the unbound 5''-MD enzyme activity was measured in the supernatant using [125I]rT3 in the presence of dithiothreitol. Incubation with 3.2 .+-. 0.9 .mu.g (mean .+-. SEM; n = 4) rat liver SMP inhibited the binding of active 5''-MD to protein-A-bound antibody by 50%. Based on doses with similar 50% binding inhibitory activity, liver SMP were 2.3 times more potent than liver microsomes and 19 times more potent than liver homogenate; liver nuclei and mitochondria showed little or no inhibition of the binding of 5''-MD to antibody. The relative 5''-MD content of liver, kidney, pituitary, placenta, and cerebral cortex based relative potency in the MBIA approximated 100:25:8:3:3. The threshold of the MBIA approximated 0.9 .mu.g liver SMP/tube. The coefficient of variation approximated 2% within an assay and 6% between assays. To obtain a cDNA clone for 5''-MD we used the SMP antibody to screen a rat liver .lambda.gt11 cDNA expression library. Of 16 positive (antibody-reactive) clones that were isolated, the fusion proteins from only 2 (no. 23 and 54) inhibited the binding of 5''-MD in rat liver SMP to protein-A-bound rabbit antibody in a dose-dependent manner. Western blot analysis showed that the molecular size of liver protein encoded by clones 23 and 54 approximates 31K. Sequence analysis showed that clone 23 insert is 804 basepairs long, contains a single long open-reading frame, and is identical to clone 54. Southern blot analysis showed that clones 23 and 54 did not cross-hybridize DNA from other SMP antibody-positive clones. Northern blot analysis using clone 23 insert cDNA as the probe showed a hybridization band corresponding to a mRNA approximating 2.8 kilobases. The expression of mRNA for the putative 5''-MD gene and type I 5''-MD activity were similarly affected by thyroid dysfunction in liver and were similarly distributed among liver, kidney, cerebral cortex, and brown fat tissues. We conclude that the MBIA is a useful method to identify 5''-MD (catalytically active or inactive) immunologically, clones 23 and 54 encode at least a portion of 5''-MD, the MBIA should be useful in further studies of the 5''-MD protein, and the cDNA clones will enable study of the gene(s) from which it is synthesized.